Method of potentiating acth preparations



United States Patent 2,992,165 METHOD OF POTENTIATING ACTH PREPARATIONS Robert E. Thompson, 204 E. Third St., Momenc'e, Ill. No Drawing. Filed Oct. 1, 1957, Ser. No. 687,344 11 Claims. (Cl. 167-74) This invention relates to a method of potentiating ACTH (corticotropin). More particularly, the invention is concerned with a method of enhancing the ACTH activity demonstrated on subcutaneous administration of an ACTH preparation.

ACTH preparations are usually administered either intravenously or extravascularly (subcutaneously and/or intramuscularly). The corticotropin assay procedures set forth in the United States Pharmacopeia WI (U.S.P.) relate to the mode of administration, the intravenous (I.V.) rat assay being used for establishing the potency of an ACTH preparation on intravenous administration, and the subcutaneous (S.C.) rat assay being used to determine the potency of an ACTH preparation on subcutaneous or intramuscular administration. These assays depend on the depletion in ascorbic acid content of the adrenal gland in hypophysectomized rats. Each of the assays has been found to correlate quite closely with clinical experience for the corresponding routes of administration. However, the intravenous potency of an ACTH preparation is usually substantially different than its subcutaneous potency as determined by the respective U.S.P. rat assays. ACTH preparations have been tested which have a ratio of subcutaneous potency to intravenous potency from less than 1:1 to greater than 3:1.

The general object of this invention is to provide a method of potentiating the U.S.P. subcutaneous units of an ACTH preparation. A related object is to provide a method of accomplishing the stated result without at the same time substantially decreasing the U.S.P. intravenous units of the preparation. Another object is to provide a method of increasing the ratio of subcutaneous to intravenous potency of an ACTH preparation. A further object is to provide a method of recovering a higher yield of SC. units from the pituitary gland source material.

The present application is a continuation-in-part of my co-pending application Serial No. 547,529, filed November 17, 1955, now abandoned, for Enhancement of Adrenoco-rticotrophin Potency.

In accordance with the present invention, an aqueous solution of an ACTH-active material is heated at a pH of from 5.5 to 8.0 and at a temperature of from 50 to 150 C. It is known that ACTH is subject to oxidative inactivation, this destructive reaction being promoted by a neutral pH and heat. It is therefore surprising to find that such pH and temperature conditions favor a reaction which potentiates the subcutaneous potency of an ACTH preparation. In order to achieve the full benefits of this potentiation reaction, however, it is desirable to carry out the heating step while inhibiting the oxidation of the ACTH. Also, the ACTH materal being treated is preferably of a particular character, as will subsequently be described.

While some benefits can be obtained by applying the method of this invention to any ACTH-active material, it is preferred to select a material for treatment which has a subcutaneous potency at least equal to but not over. 3.5 times its intravenous potency, as measured respectively by the U.S.P. subcutaneous and intravenous rat assays. Still better results can be obtained with an ACTH-active material demonstrating a ratio of subcutaneous to intravenous potency of from about -1 to 3, and an especially desirable ACTH-active material to be subjected to treatment according to this invention demonice strates a ratio of subcutaneous to intravenous potency of from 1 to 2. While most ACTH extracts and concentrates fall into this category, there are exceptions, such as a fully potentiated ACTH material produced by the method of the present invention. In other Words, for any ACTH preparation, there is apparently a maximum level of potentiation of subcutaneous activity in relation to intravenous activity. Most preparations can be potentiated to a ratio of subcutaneous to intravenous potency of greater than 3:1, and many preparations to subcutaneousintravenous potency ratios of 4:1 or 5:1.

the degree of purification of the particular ACTH preparation, since both measures of potency by the U.S.P.-

tests are in units per milligram. Consequently, the method of this invention must be distinguished from strictly purification procedures in which the I.V. units and the 5.0. units per milligram are both increased without their ratios of one to the other being substantially altered.

The method of this invention in certain of its embodiments is illustrated by the following examples.

Example I To 20 ml. of an aqueous solution of porcine ACTH prepared by glacial acetic acid extraction and oxycellulose purification was added 200 mg. of l-cysteine base. The resulting solution was adjusted to a pH of 7.4 with a 10% solution of sodium hydroxide. This pH adjustment resulted in the formation of a precipitate in the solution. This mixture was filled into a glass vial, and the filled vial was sealed with a rubber stopper and an aluminum seal.

The sealed vial was immersed in a boiling water bath for incubation. Portions of the vial contents were removed at selective time intervals. The portions were combined with 16% aqueous gelatin containing 0.5% of phenol for assay by the U.S.P. subcutaneous assay procedure.

The average incubation temperature was to C. The assay results were as follows:

Time interval (minutes): units per ml.)

These results indicate that maximal enhancement of the subcutaneous potency of porcine ACTH prepared by' glacial acetic acid extraction and oxycellulose purification can be achieved in about two hours at a temperature of 90 to 100 C., and that thereafter the activity fallsoff at a rapid rate.

Example 11 To 40 ml. of an aqueous solution of porcine ACTH prepared by glacial acetic acid extraction and oxycellulose purification was added 200 mg. of l-cysteine base. The resulting solution was adjusted to a pH of 6.5 with a 10% solution of sodium hydroxide, whereupon a precipitate was formed in the solution.

-A portion of this mixture, 4 ml., was filled into a 5 ml. glass vial. The air in the filled vial was displaced with nitrogen gas, and the vial was sealed.

The filled vial was introduced into an oven at a temperature of 100 C. Portions of the vial contents were removed at selected time intervals during incubation.

These portions were combined with 16% aqueous gelatin Patented July. 11,

It will be understood that these potency ratios are independent of" Potency U.S.P.-s0.

3 containing 0.5% of phenol for assay by the U.S.P-S.C. procedure;

The results were as follows:

Potency (U.S.P.-SC.

Timeinterval (minutes): units per ml.)

Exemple III Crude porcine ACTH obtained by glacial acetic acid extraction in the amount of 210 mg. was dissolved in 30 ml. of water adjusted to pH 4.0 with acetic acid. To the resulting solution was added 150 mg. of l-cysteine hydrochloride, and this solution was adjusted to pH 7.3 with a solution of sodium hydroxide. Thereupon a precipitate formed in the solution.

This mixture was filled into a glass vial, and the filled vial was sealed. The sealed vial was incubated at a temperature of 90 to 100 C., and portions of the vial contents were removed at selective time intervals during incubation. These portions were combined with 16% aqueous gelatin containing 0.5% of phenol for assay by the U.S.P-S.C. procedure.

The results were as follows:

Potency (U.S.P.-SC.

units per'mg.)

Time interval (hours) Example IV Bovine ACTH (prepared by acid acetone extraction and oxycellulose purification) in the amount of 210 mg., was dissolved in 30 ml. of water adjusted to pH 4.5 with acetic acid. To the resulting solution was added 150 mg. of l-cysteine hydrochloride. This solution was adjusted to; pH 7.2 with a 10% solution of sodium hydroxide, whereupon a precipitate formed in the solution. This mixture was filled into a glass vial, and the filled vital was sealed.

I The sealed vial was incubated at a temperature of 90 to 100 C., and portions of the vital contents were removed at selected time intervals during incubation. These portions were combined with 16% aqueous gelatin containing 0.5 of phenol for assay by the U.S.P-S.C. procedure.

The results were as follows:

Potency (U.S.P.-SC.

Time interval (hours): units per mg.)

These results indicate that maximal enhancement of subcutaneous potency can be obtained with bovine ACTH prepared by acid acetone extraction and oxycellulose purification in about 16 hours at a temperature of 90 to 100 C. and a pH of 7.2.

Example V Bovine ACTH (prepared by acid acetone extraction and oxycellulose purification), in the amount of 60 mg.,

. filled vials were sealed. The sealed vials were incubated at a temperature of 100 C., and portions of the vial contents were removed at selected time intervals for assay by the U.S.P.-SC. procedure.

The results were as follows:

Potency (U.S.P.-SC.

Time interval (hours): units per mg.)

These results indicate that when gelatin and phenol are included in the incubation mixture an increase in the rate of enhancement for bovine ACTH prepared by acid acetone extraction and oxycellulose purification is obtained at a particular temperature.

Example VI Bovine ACTH (prepared by acid acetone extraction and oxycellulose purification) Was dissolved in a 0.1% acetic acid solution to obtain an aqueous solution containing 10 mg. per cc. of ACTH. To this solution was added 0.5% of l-cysteine hydrochloride and the resulting solution adjusted to a pH of 6.9 with a 10% solution of sodium hydroxide. The resulting suspension was filled into a glass vial, and the air in the vial was displaced with nitrogen gas. The filled vial was sealed, and incubated at a temperature of to C. for a period of 11 hours.

The incubated suspension was cooled to a temperature of 30 to 50 C., and then strained through nylon cloth. The filtrate was combined with a 16% aqueous gelatin solution containing 0.5% of phenol. To this gelatin solution was added 0.1% of l-cysteine hydrochloride.

The gelatin solution was clarified by filtration, vialed under nitrogen, and autoclaved for 20 minutes at a pressure of 15 p.s.i.g. and a temperature of C.

In the preparation of this product a pilot curve was obtained by removing portions of the vial contents at selected time intervals during incubation. These portions were assayed by the U.S.P.-SC. procedure. The results of the pilot curve were as follows:

Potency (U.S.P.-SC.

Time interval (hours) units per mg.)

4 20 (one level assay).

8 31 (one level assay).

12 35.1145 (three level assay). 16 21 (one level assay).

On the basis of this pilot curve a time interval of 11 hours was selected for incubation of the master batch. The finished product was assayed by the U.S.P.-SC. procedure, and, in a three level assay, was found to contain 300:2.7 units per mg. or ml.

The separated precipitate was obtained in a yield of 40 mg., and was found by the U.S.P.-SC. procedure to contain less than 5 U.S.P. units per mg.

These results indicate that a bovine ACTH completely soluble in water at the neutral pH range can be obtained cWith enhanced subcutaneous potency. The insoluble residue separated in the process was found to contain negligible ACTH activity.

Example VII Porcine ACTH obtained by acid acetone extraction and oxycellulose purification was mixed 10 rug/m1. with 16% gel-0.5% phenol. The pH was adjusted to 6.5.

The liquid was placed in a sealed container and heated at 95-100 C. for 12 hours. Samples were withdrawn at 0, 4, 8 and 12 hours and assayed subcutaneously in gelatin-phenol. Assay results may be tabulated as follows:

Time at 100 C.: S.C. units/mg. ACTH hours 40:4 4 hours 54:6 8 hours 63:7 12 hours 34:4

Under the above conditions, it is shown that the subcutaneous potency increases to a maximum of about 150% of the starting potency at about 8 hours.

Example VIII 100 grams of porcine ACTH obtained by acid acetone extraction and oxycellulose purification was mixed with 20 liters 16% gelatin containing 0.5% phenol. The pH was adjusted to 6.5 with a 20% solution of sodium hydroxide. The preparation was placed in a stainless steel container, sealed and autoclaved at 115-121 C. for 20 minutes. The autoclaved material was diluted to 120 liters with 16% gelatin containing 0.5% phenol. The liquid was clarified through Ertel No. S pads, and then sterile filtered through E-9 pads. After holding the bulk overnight, it was filled into approximately 20,000 x ml. vials, sealed as usual, and autoclaved at 115-121 C. for 20 minutes. This procedure was repeated on each of four lots of comparable ACTH in gelatin. The original ACTH assayed 31:3 S.C. units per mg. Assay of finished product showed a recovery of 47:25 S.C. units per mg. (average of four lots). This shows a recovery of about 150% of the starting potency before autoclaving in gelatin-phenol at pH 6.5.

Example IX Porcine ACTH obtained by acid acetone extraction and oxycellulose purification was mixed 10 m-g./ml. in 16% gelatin containing 0.5% phenol. 0.4% l-cysteine was added, and the pH was adjusted to 6.5 with a 20% solution of sodium hydroxide. The preparation was placed in a sealed container under nitrogen and heated at 95-100 C. for 12 hours. Samples were withdrawn and assayed at 0, 4, 8 and 12 hours by the usual procedure, subcutaneously. Results are tabulated as follows:

Time at 100 C.: S.C. units per mg. ACTH 0 hours 40:4 4 hours 80:8 8 hours 100:12 12 hours 80:10

Thus it can be seen that through the cooperating effect of gelatin or gelatin-phenol, near neutral pH, l-cysteine and heat, a dramatic increase in subcutaneous potency is obtained, i.e. an increase in potency of about 250% at the optimal point of 8:4 hours at 95 to 100 C.

Example X 200 mg. porcine ACTH (31:3 S.C. and 21:1.V. units/mg.) was mixed with 10 ml. gelatin-phenol. 0.5% l-cysteine was added. The pH was adjusted to 6.5, and the liquid was vialed under nitrogen and heated for 5V2 hours at 95100 C. Assay of the finished material showed 65:6 S.C. units per mg.

Example XI 75 grams of acid acetone extracted, oxycellulose purified porcine ACTH (31:3 S.C. and 21:2 I.V. u./mg.) is mixed with 3.75 liters water. Then 3.75 liters 32% gel containing 1% phenol is added and mixed well. 0.3% l-cysteine is added and the pH is adjusted to 6.5 with a 20% solution of sodium hydroxide. This mixture is placed in a sealed container under nitrogen and heated in a stream autoclave at 95-100 C. for 5 hours. After removal from the autoclave, the liquid is filtered through a sintered glass filter to remove about 6-10 grams (dry Weight) of a neutral insoluble fraction. This fraction contains less than 2% of the total activity of the batch.

The pH of the 15 liter bulk is readjusted to 6.5 with a 20% solution of sodium hydroxide. Then the material is replaced in a sealed container under nitrogen and autoclaved at -121 C. for 20 minutes and then immediately cooled to room temperature. The bulk is then sterile and may be stored at room temperature or in a refrigerator for months Without deterioration.

The 15 liters of autoclaved bulk is diluted to -130 liters (depending on bulk assay) with 16% gelatin containing 0.5 phenol. The pH is adjusted to 6.4, and the liquid is filtered through E-8 or E-9 pads, vialed under nitrogen. The finished vials are autoclaved at 115-121 C. for 20 minutes for sterilization.

Each heat step above contributes to total heat input to approximate the 8 hour-100 C. indicated in previous examples as optimal for this ACTH.

In the foregoing examples, reference has been made ta ACTH prepared by glacial acetic acid extraction and oxycellulose purification. The extraction procedure for such ACTH preparations is described in J. Biol. Chem., vol. 187, pages 719-731, and the purification procedure is described in J. Am. Chem. Soc., vol. 73, pages 2969- 2970. Such ACTH preparations will usually be suitable for treatment by the method of the present invention. It has been found, however, that the ratio of S.C. to I.V. activity is subject to considerable variation from batch to batch, and that the total recovery of S.C. units is also subject to variation. Consequently, it is preferred to use as a starting material for the present process an ACTH preparation which has been obtained by an acid acetone extraction, either before or after oxycellulose purification. With ACTH preparations obtained by acid acetone extraction, there is substantially less variability in the ratio of S.C. to I.V. activity. Further, a substantially greater yield of ACTH activity in terms of S.C. units can be obtained so that after the product is potentiated by the method of the present invention, the total yield of S.C. units may be as much as 100% greater than with a product obtained by glacial acetic acid extraction, either with or without oxycellulose purification.

One preferred procedure for producing a starting material for the present invention is as follows: Fresh whole hog pituitaries are comminuted with Dry Ice. The milled glands are then extracted with a mixture of acetone and hydrochloric acid (78-80% acetone, pH 1.5). After separation of the extract from the tissue residue, the extract is chilled and 100% acetone is added to bring the acetone concentration of the extract to about 90-92%. The precipitate thus formed is separated, washed and dried. The product, sometimes referred to as acid acetone powder, can then be potentiated by the method of this invention, or it can be subjected to oxycellulose purification prior to potentiation.

As indicated previously, it is preferred to carry out the potentiating treatment of the present invention while inhibiting the oxidation of the ACTH. The simplerneans of accomplishing the desired result is to include a reducing agent in the aqueous solution. Cysteine has been found to give particularly good results in controlling the destructive oxidation of ACTH, but other organic and inorganic reducing agents can be used. A preferred class is the organic reducing agents having equivalent properties to cysteine in that they contain a sulfhydryl group. Among the reducing agents of this type are hydrogen sulfide, beta mercaptoethanol, thioglycollic acid, etc. A closely related organic reducing agent is thiourea. Other agents of value in controlling the oxidative destruction of ACTH are inhibitors like glycine and 8-hydroxyquinoline.

As another means of inihibiting the oxidation of ACTH, the heating procedure can be carried out in the absence of oxygen; For example, the atmospheric air can be dis-- placed with nitrogen, or the heating can be carried out under an atmosphere of a reducing gas like hydrogen. Suchprocedures can be used as an adjunct to the incorporation of a reducing agent or oxidation inhibitor in the aqueous ACTH solution. If the oxidation inhibition is insufiicient and ACTH activity is being destroyed, this will show up by the reduction of I.V. activity. In other words, the progress ofthe potentiation reaction can be followed by the increase of S.C. activity, while the undesirable destructive reaction can be followed by determining whether any loss of I.V. activity has occurred. Obviously, some loss of activity may occur in conjunction With a greater increase in subcutaneous activity due to the potentiation reaction, thereby resulting in a net increase of S.C. activity. Under the most desirable conditions, however, every precaution is taken to minimize the inactivation while maximizing the potentiation.

It has been found that-the signficant degree of potentiation of S.C. units and the greatest over-all yield of S.C. units is obtained when the starting material has a U.S.P. subcutaneous potency ranging from one to two times its U.S.P. intravenous potency. A typical starting material of this type would be an acid acetone extract, oxycellulosepurified material having a ratio S.C. to I.V. potency of approximately 1.5: 1. With such a starting material as well as with other ACTH-active materials, the preferred pH of treatment is from about 6.2 to 7.5 and the preferred temperature is about 75 to 125 C. According to the method of this invention, the heating is continued until the S.C. potency has been increased, or preferably until the ratio of S.C. to I.V. potency is substantially over 3:1.

When gelatin is present during the heat potentiation, as

preferred, it is possible to remove any insoluble residue 7 which is formed without substantial loss of ACTH activity. The gelatin acts as a solubilizing agent for the ACTH so that no substantial amount of activity is absorbed when the insoluble protein precipitates. Thus, a completely soluble product at a neutral pH having a maximal S.C. activity can be produced.

While in the foregoing specification this invention has been described in relation to a number of specific embodiments thereof and many details have been set forth for purpose of illustration, it will be readily apparent to those skilled in the art that the invention is susceptible to additional embodiments and that many of the details can be varied considerably without departing from the basic concept of the invention.

I claim:

1. Method of potentiating U.S.P subcutaneous units of an ACTH preparation, comprising heating an aqueous solution of ACTH-active material having a pH of from 5.5 to 8.0' at a temperature of from 50 to- 150 C. while inhibiting the oxidation of the ACTH to significantly enhance the subcutaneous potency of said ACTH-active material, said ACTH-active material prior to heating having a subcutaneous potency of at least equal to but not over 3.5 times its intravenous potency, as measured respectively by the U.S.P. subcutaneous and intravenous rat assays.

2. The method of enhancing the ACTH activity of a corticotropin preparation demonstrated on subcutaneous administration, which comprises heating an aqueous solution of gelatin, a reducing agent and an ACTH-active ma terial derived from porcine pituitary tissue having a pH of from 5.5 to 8.0 at a temperature of from 50 to 150 C. to significantly enhance the subcutaneous potency of said. ACTH-active material, said ACTH-active materialprior to heating having a ratio of subcutaneous to intravenous potency as measured by the U.S.P. assay procedures of froml to 3.5.

3. The method of claim 2 in which said reducing agent is an organic reducing agent containing at least one sulfhydryl group.

4. The method of claim 2 in which said reducing agent is cysteine;

5; The method of enhancing the ACTH activity of a corticotropin preparation demonstrated on subcutaneous administration, which comprises heating an aqueous solution of a reducing agent and an ACTH-active material derived from bovine pituitary tissue having a pH of from 5.5 to 8.0 at a temperature of from 50 to 150 C. to significantly enhance the subcutaneous potency of said ACTH-active material, said ACTH-active material prior to heating having a ratio of subcutaneous to intravenous potency as measured by the U.S.P. assay procedures of from 1 to 3.5.

6. The method of claim 5 in Which said reducing agent is an organic reducing agent containing at least one sulfhydryl group.

7. The method of claim 5 in which said reducing agent is cysteine.

8. The method of potentiating U.S.P. subcutaneous units of an ACTH preparation, comprising heating an aqueous solution of a substantially unhydrolyzed, ACTH- active material having a pH of from 5.5 to 8.0 at a temperature of from to C. While inhibiting the oxidation of the ACTH to enhance the subcutaneous potency of said ACTH-active material to substantially more than 3 times its intravenous potency, said ACTH-active material prior to heating having a subcutaneous potency at least equal to but not over 3 times its intravenous potency, as measured respectively by the U.S.P subcutaneous and intravenous rat assays.

9. The method of claim 8 in which said ACTH-active material is obtained by extracting ACTH-containing pituitary tissue with acidified acetone.

10. The method of claim 8 in which said ACTH-active material has a ratio of subcutaneous to intravenous potency as measured by the U.S.P. assay procedures of from 1 to 2.

11. The method of potentiating U.S.P. subcutaneous. units of an ACTH preparation, comprising heating an aqueous solution of a reducing agent and a glacial acetic acid-extracted, oxycellulose-purified ACTH-active material having a pH of from 5.5 to 8.0 at a temperature of from 75 to 125 C. to enhance the subcutaneous potency of said ACTH-active material to substantially more than 3.5 times its intravenous potency, said ACTH-active material prior to heating being substantially unhydrolyzed and having a subcutaneous potency at least equal to but not over 3.5 times its intravenous potency, as measured respectively by the U.S.P. subcutaneous and intravenous rat assays.

OTHER REFERENCES Farmer: Biochem. 1., vol. 59, 1955, p. XII. Dixon: Biochem. 1., vol. 61, 1955, p. 483, 493. Collip: Symposia, Quant. Biol., vol. 5, 1937, p. 212. 

1. METHOD OF POTENTIATING U.S.P SUBCUTANEOUS UNITS OF AN ACTH PREPARATION, COMPRISING HEATING AN AQUEOUS SOLUTION OF ACTH-ACTIVE MATERIAL HAVING A PH OF FROM 5.5 TO 8.0 AT TEMPERATURE OF FROM 50 TO 150*C. WHILE INHIBITING THE OXIDATION OF THE ACTH TO SIGNIFICANTLY ENHANCE THE SUBCUTANEOUS POTENCY OF SAID ACTH-ACTIVE MATERIAL, SAID ACTH-ACTIVE MATERIAL PRIOR TO HEATING HAVING A SUBCUTANEOUS POTENCY OF AT LEAST EQUAL TO BUT NOT OVER 3.5 TIMES ITS INTRAVENOUS POTENCY, AS MEASURED RESPECTIVELY BY THE U.S.P. SUBCAUTANEOUS AND INTRAVENOUS RAT ASSAYS. 